In this reporting period we published initial findings from the intramural NINDS PDRisk study and introduced novel biomarkers of catecholaminergic neurodegeneration. (1) Biomarkers of cardiac noradrenergic and central dopamine deficiency predict Parkinson disease (PD) in at-risk individuals: We have found that among people at risk for developing PD those who develop symptomatic disease within 3 years have at entry neuroimaging evidence of cardiac noradrenergic deficiency, identified by low myocardial 18F-dopamine-derived radioactivity (Goldstein et al., Parkinsonism Relat Disord. 2018;52:90-3), and low cerebrospinal fluid (CSF) levels of both 3,4-dihydroxyphenylacetic acid (DOPAC, the main neuronal metabolite of dopamine) and 3,4-dihydroxyphenylalanine (DOPA, the precursor of dopamine) (Goldstein et al., Park Rel Dis 2018; 50:108-112). (2) Extending the PDRisk cohort in a new clinical protocol: Under a recently approved protocol (NIH Clinical Protocol 18-N-0140, Clinical Laboratory Evaluation of Chronic Autonomic Failure) we are extending the PDRisk cohort. (3) A quantitative biomarker of alpha-synuclein deposition in sympathetic noradrenergic nerves: AS deposition in sympathetic noradrenergic nerves characterizes Lewy body forms of neurogenic orthostatic hypotension (nOH). Using immunofluorescence confocal microscopy for AS and tyrosine hydroxylase (an index of sympathetic noradrenergic innervation) we described qualitatively a spectrum of abnormalities of ganglionic AS and TH in chronic autonomic failure syndromes (Isonaka et al., Clin Auton Res 2018;28:223-230; Isonaka et al., Clin Auton Res 2017;27:97-101; Goldstein et al., Clin Auton Res 2017;27:57-62). We validated a quantitative index of AS colocalization with TH in post-mortem sympathetic ganglion tissue and are applying this colocalization index clinically in skin biopsies obtained under NIH Clinical Protocols 03-N-0004 and 18-N-0140. Preliminarily, values for the AS/TH colocalization index in sympathetic noradrenergically innervated skin constituents completely separate Lewy body from non-Lewy body nOH. (4) A potential clinical biomarker of spontaneous oxidation of dopamine in the human brain: 5-S-Cysteinyldopamine (Cys-DA) is a product of the spontaneous oxidation of cytoplasmic dopamine. Our preclinical studies have shown that inhibiting enzymatic dopamine oxidation via a monoamine oxidase inhibitor (MAOI) increases spontaneous dopamine oxidation as indicated by increased Cys-DA levels (Goldstein et al., J Pharmacol Exp Ther 2016;356:484-493; Goldstein et al., Neurochem Res 2016;41:2173-2178; Goldstein et al., Neurochem Res 2017;42:3289-3295). Based on our clinical finding that human CSF normally contains Cys-DA (Goldstein et al., Park Rel Dis 2016;31:79-86), under NIH Clinical Protocol 17-N-0076 we are assessing whether antioxidation with N-acetylcysteine (NAC) decreases CSF Cys-DA in PD patients on a MAOI. (5) Collaborations: (a) Relationship of AS deposition in pilomotor muscles with cardiac noradrenergic deficiency in autonomic synucleinopathies: Under NIH Clinical Protocol 03-N-0004 and an approved Material Transfer Agreement we provided skin biopsies from patients with Lewy body nOH or MSA to collaborators at Harvard (Roy Freeman, Christopher Gibbons). AS was measured in pilomotor muscles, which receive pure noradrenergic innervation. AS content was adjusted for local sympathetic innervation by calculating the ratio of AS to PGP 9.5, a pan-axonal marker. The Lewy body group had higher AS/PGP 9.5 ratios and lower concentrations of 18F-dopamine-derived radioactivity in the left ventricular myocardial than did the MSA group. These preliminary data indicate that among patients with nOH elevated AS/PGP9.5 ratios in pilomotor muscles and low cardiac 18F-dopamine-derived radioactivity efficiently distinguish Lewy body nOH from MSA. (b) AS deposition in submandibular gland, myocardium, and skin in PD+OH: In a collaborative study with Thomas Beach (Banner Sun Health Research Institute), under a Material Transfer Agreement we plan to assay post-mortem submandibular gland, myocardium, and skin samples from patients with PD+OH and controls, as an extension of our research on AS/TH colocalization in sympathetic noradrenergically innervated tissues. (c) Intramural collaborative studies of genotype-phenotype relationships in familial forms of catecholaminergic neurodegeneration: We reported previously that in rare cases of familial PD due to mutation or triplication of the AS gene the genotypic abnormalities cause sympathetic neurocirculatory failure and decreased vesicular sequestration in residual noradrenergic terminals. Extending on these findings, we are collaborating with intramural NIH investigators (Derek Narendra and Sonja Scholz, NINDS; Ellen Sidransky, NHGRI) to explore genotype-phenotype relationships in other familial forms of PD, including Gaucher/PD, AS gene duplication, and mutation of the genes encoding DJ-1 or parkin. (d) Biomarkers research in the Autonomic Rare Diseases Clinical Research Consortium (RDCRC): As a member institution of the Autonomic RDCRC we shared data from patients with nOH in a study on the natural history of PAF (Kaufmann et al., Ann Neurol 2017;81:287-297; Norcliffe-Kaufmann et al., Ann Neurol 2018;83:522-531), provided skin biopsy specimens from patients with nOH for analysis of AS deposition (see (a) above), and assayed CSF catechols as part of an experimental therapeutic trial of mesenchymal stem cells for MSA (Project No. ZIA NS003125). (e) Clinical catecholamine neurochemistry in chronic fatigue syndrome: We are collaborating in an intramural study of chronic fatigue syndrome (NIH Clinical Protocol 16-N-0058, Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome, PI Avindra Nath) by conducting screening autonomic function testing and provocative tilt table testing and assaying plasma and cerebrospinal fluid levels of catechols. (f) Clinical catecholamine neurochemistry in postural tachycardia syndrome (POTS): We conducted a collaborative study with Satish Raj (Vanderbilt) in which we assayed plasma and urine catechols from a large cohort of patients with POTS and control subjects, with the goal of identifying a POTS subgroup with neurochemical evidence of decreased activity of the cell membrane norepinephrine transporter. (g) Clinical catecholamine neurochemistry in spinal cord injury (SCI): Patients with high level SCI provide a model of disruption of sympathetic noradrenergic outflows with intact parasympathetic cardiovagal outflow. We continued a collaborative study with Jill Wecht (Bronx VA Hospital), in which we are assaying plasma catechols in patients with chronic SCI, especially to assess orthostatic reflexive activation of the sympathetic noradrenergic and sympathetic adrenergic systems. (h) Clinical catecholamine neurochemistry in stroke from intracranial bleeding: In a collaborative study with Oladi Bentho (Univ. of Minnesota), we assayed plasma catechols in patients with acute stroke from intracranial bleeding, to determine if plasma catecholamines provide prognostic biomarkers.